Occurrence of (20R)- and (20S)-Δ8(14) and Δ14 5α(H)-sterenes and the origin of 5α(H),14β(H),17β(H)-steranes in an immature sediment

~16~7037/89/$3.~

Gsoclrimicaa Cosmochimica Acta Vol.53,pp.2001-2009

Copyright B 1989P-on

+ .30

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and Al4Sa(H)-sterenes and the origin of Occurrence of (20R)and (20S)-A8t14) 5a(H),l4~(H),l7~(H~steranes in an immature sediment* J. R. RECHKA’, J. W. DE LEEUW*and J. R. MAXWELL’ T. M. PEAKMAN”*‘~,H. L. TEN HAVENING, ‘Organic Geochemistry Unit, School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 lTS, U.K. *Organic Geochemistry Unit, Department of Chemical Technology and Materials Science, Delhi University of Technology, De Vries van Heystplantsoen 2,2628 RZ Del&, The Netherlands 31nstitute of Petroleum and Organic Geochemistry (ICH-51, KFA Jiilich, P.O. Box 19 13, D-5 170 Jiilich 1, Federal Republic of Germany (Received November 8, 1988; accepted in revisedform May 5, 1989)

Abstract-Series of (20R)- and (20S)-Ah8(‘4)and Al4 Sor(H)-sterenes have been identified in an immature marl from a hypersaline deposit (northern Apennines, Italy) by co-injection studies with authentic compounds. These alkenes co-occur with A 13(17)Sa(H)-spirosterenes and transient (2OR)- and (2OS)-Al4 SLY(H),17/3(H)-sterenes. The source of these isomeric steroid alkenes is probably A’ (and Asci4))5&(H)sterols via (A**‘) A2*q’4)and Az,14S~(H~stemdienes. Reduction processes in the sediment, via the Al4 5~(H),l7~(H)-st~~n~, can give rise to the C-20 isomeric 5~(H),i4~(H),l7~(H~ste~n~ present in “anomalously” high relative abundances from a thermal maturity viewpoint. This diagenetic pathway is supported by laboratory rearrangement/hydrogenation experiments of Sa(H)-cholest-7-ene and from considerations of carbon number distributions of various steroid hydrocarbons in the sediment extract. carbon number homologues of various classes of steroid hy-

General STERANEsARECOMMONconstituents of sedimentary organic matter. Two major series have been recognised, the so-called regular series and the diasterane series. Both occur as a range of carbon numbers (mainly C+&, although in certain environments C2, , C2, , CZ6and Cm can aIso be impo~nt) and as mixtures of stereo-isomers. The degree of isomerisation, at positions 14, 17 and 20, within the regular series has been widely applied to assess the maturity of source rocks and petroleum (e.g., SEIFERT, 1980; SEIFERT and MOLDOWAN, 198 1; MACKENZIEet al., 1980; MACKENZIE, 1984) and, in conjunction with other biological marker parameters, to attempt to understand and reconstruct the thermal and burial history of sediments (e.g., MACKJZNZ~E and MCKENZIE, 1983; BEAUMONTet al., 1985), and generation history of crude oils and asphalts (RULLKOTTERet al., 1985, 1986). It is now becoming apparent that there can be maturity discrepancies in the sterane distributions in certain immature sediments (TEN HAVEN et al., 1985, 1986, 1988), notably in the relative abundance of (20& and (20S)-5@(H),14&H), 17@(H)-isomers. These components have been suggested as arising from A’ Srr(H)-sterols via A’, As(‘4tand Al4 5cY(H)-sterenes and A”(“) Sa(H)-spirosterenes (TEN HAVEN et al., 1986; PEAKMANand MAXWELL, 1988a). In addition, another recent study (Cuu, 1987) has indicated apparent “anomalies” between extents of isomerisation within the * Part 111in the series “Organic geochemical studies of a Messinian evaporitic basin, northern Apennines (Italy)“. This work was presented in part at the 192nd ACS National Meeting. Anaheim. Seotember 1986 (Abstract No. 48). For Part II see SIN%NGHE D&& et aI. (1986). t Corresponding author. Present address Institute of Petroleum and Organic Geochemistry (ICH-.5), KFA Jiilich, P.O. Box 1913, D5 170 Jiilich 1,Federal Republic of Germany. 2001

drocarbons. In this paper we have reexamined the distributions of steroid hydrocarbons in a marl from the northern Apennines (cj, TEN HAVEN et al., 1985) to investigate in more detail recent hypotheses (TEN HAVEN et al., 1985, 1986; CURIALE, 1987; PEAKMAN and MAXWELL,1988a) concerning the lowtemperature diagenesis of steroids which can lead to “anomalies” in steroid maturity indicators. We have also carried out additional rearrangement/hydrogenation experiments of A7 Sa(H)-sterenes to investigate their proposed transformation to (2OR)- and (20S)-5a(H), 14/3(H),17@(H)-steranes via A’3(‘7) So!(H)-spirosterenes (TEN HAVEN et al., 1985, 1986; PEAKMAN and MAXWELL, 1988a). The various steroid skeIetal types discussed are surnrn~~ in Fig. 1 and a list of the specific compounds is given in Table 1. Steroids related to the regular series have 8/.?(H),9o(H), lO@(Me), 13P(Me), 14a(H), 17a(H)-stereochemistry, whilst dia- or rearranged-steroids have 5a(Me),ga(H),g@H), 14@(Me>stereochemistry, unless otherwise stated. Steroid diagenesis The steranes of relatively immature sediments consist predominantly of (2OR)&(H), 14a(H), 170(H)- and (20&58(H), 14c~(H),17rr(H)-components in a typical ratio ofcu. 3-4: 1 (e.g., MACKENZIE, 1984).They are believed to arise from reduction of the corresponding sterenes (principally A*, (A3), A” and A’), which in turn arise from the oxygenated steroids of organisms (reviewed by MACKENZIE et al., 1982; DELEEUWand BAAS,1986). Recently it has been suggested that A2 and proposed A’ sterenes arise from 5ru(H)-stanols and S@(H)-stanols, respectively, and hence have the corresponding stereochemistry at C-5 (DE LEEUWet al., 1989). Additionally, migration of the A2 double bond to A4 and A5 is now considered unlikely since double bond isomerisations during early diagenesis are thought to occur via tertiary and not via secondary carbonations. The A5 sterenes appear to arise, therefore, from preferential reduction of the A3double bond of A3*5 steradienes (DE Lmuw et al.,1989) which in turn arise from dehydration of A5 sterols

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T. M. Peakman et ai

R STEROID

(TYPE

DIASTEROID

I)

(‘NPE

II)

R

& H

H

PARTMLLY

SPIROSTERENE

R-H. R’=H,

H

REARRANGED

(TYPE IV)

STEROID

(TYPE

III)

REARRANGED

SPIROSTERENE (lYPEV)

Me Me, Et

FIG. I. General structural types of compounds discussed.

1982; WAKEHAM et al., 1984). A5 sterenes are isomer&d in the laboratory under mild acid conditions to an equilibrium mixture of A4 and A5 components; (6 1:39 in favour of the A4 sterenes; KIRK and SHAW, 1975). A similar isomerisation is presumably operating in sediments since this occurs via the C-S tertiary carbocation (DE LEEUW ef al., 1989, TEN HAVEN et al., 1989). In addition to sedimentary reduction processes, A4and As sterenes can also undergo acid-cat&& backbone rearrangement to Aryt7) lOcu(H)-diasterenes (RUBINSTEIN et al., 1975). The rearrangement has received detailed laboratory studies and it is clear that the initially formed (ZOR>isomer undergoes isomerisation at C-20 until an essentially 1:1 mixture of (20R) and (20s) is obtained (KIRK and SHAW, 1975; AKP~RIAYE et al., 1981; RAKMAN et al., 1988). The rearrangement and isomerisation at C-20 have also been observed to occur gradually with increasing burial depth in marine sediments (BRASSELLet al., 1984; TEN HAVENet al., 1989). Minor products of the backbone rearrangement have been character&d (PEAKMANet al., 1988) and identified in immature sediments (PEAKMAN and MAXWELL,1988a). These are A’307)iO~(H~~asteren~, rearranged A’wi7ftOa( and lO~(H~spiroste~n~ and com~nents of partial backbone rearrangement. With increasing maturity the A’X17fIO&H)diasterenes are reduced to loo(H), 13@(H),l7a(H)and 1Oa(H), 13a(H), 17&H)diasteranes, with the former predominating (ENSMINGER et al., 1978). The corresponding 10/3(H)-components are expected to be formed although their presence remains to be confirmed (PEAKMANand MAXWELL,1988a). Changes within the distributions of regular sterane isomers are also observed with increasing burial depth. Thus, (20S)&(H), 14a(H), 17o1(H)-and (20R)- and (20S)-5cu(H),14~(H),l7~(H)-comportents become increasingly important. Mature sediments and crude oils typically contain, therefore, mainly (20R)- and (2OLQ %X(H),14~(H),l7ff(H~ and (2OR)-and (20~~5ff(H}, 14p(H), 178(H)steranes with the latter often predominating (e.g., SEIFERTand MOLDOWAN, 1979. 1981; SEIFERT, 1980; MACKENVE et a!., 1980; MACKENZIE,1984). These isomeric steranes have been assumed to arise via an isomerisation process at chiral centres, and isomerisation has been brought about in the laboratory by heating experiments on metal catalysts in the presence of hydrogen (PETROVet al., 1976; SEIFERTand MOLDOWAN,1979) and on mineral matrices in the presence of sulphur (ABBOTTef al., 1985). The sterane distributions observed in mature petroleum (e.g., the 700-million-year-old Siva crude oil; SEIFERT,1980) and from heating experiments (PETROv et al., 1976, SEIFERTand MOLDOWAN,1979) are basically in agreement with molecular mechanics calculations on a suite of 13 cholestane

isomers (VAN GRAASet al., 1982) with the exception ofloweramounts of 5/3(H),14/3(H),17P(H)-steranes than predicted occurring in crude oils and the heating experiments. Recent evidence (TEN HAVEN et al., 1985, 1986; PEAKMANand MAXWELL,1988a) suggests that the thermodynamically more stable So(H), 140(H), 17@(H)-stemnescan be formed in immature sediments from sterene intermediates rather then solely by an isomerisation process from (20R>5dH), 14ru(H),17ru(H)-steranes. Thus, it has been suggested (PEAKMANand MAXWELL,1988a) that 5=(H)-sterols with a double bond at A’ undergo dehydration to A’,’ 5~(H~steradienes. The ready ~some~~tion of the A’ double bond to Aao4’and Ai4 (PEAKMANand MAXWELL, 1988b) suggests. however, that LI’*““~’ and A2,14S@(H)-steradienes are likely to be more abundant than A’s’ Lx(H)-steradienes (DE LEEUWef al.. 1989). Preferential reduction of the A2double bond, which is known to be easily hydrogenated (FRIED and EDWARDS,1972) would give mainly Ano4)and Al4 Scu(H)-sterenes. In the laboratory the A8(14’ and AL4Sa(H)-sterenes have been shown to undergo rearrangement to A‘x”) Sa(H)-spirosterenes which readily isomerise at C-20 (PEAKMAN and MAXWELL. 1988b). These A’3(“) 5n(H)-spirosterenes are frequently encountered in immature sediments (PEAKMANet al., 1984; BRASSELL et al., 1984 TENHAVEN et a/., 1985, 1989; CURIALIZ,1987).Further rearrangement to transient A“’ Se(H), 17~~H~steren~ followed by ~du~on can then give rise to %(H), 14@(H),t 7~(H~stemn~ (EN HAVENet ai., 1986; PEAKMAN and MAXWELL,1988a; cJ, PEAKMANand MAXWELL,1988b). 5u(H)sterols with a double bond at A8(r4!which can also be biosynthesised, obviously also fit into the above scheme. 4-Methylsteranes and 4-methyldiasteranes have also been identified in sediments and petroleums (RURINSTEINand ALBRECHT,1975;

(MACKENZIE ez al.,

Table 1: SteEid

hydrocarbons mentioned IIIthe text.

No. Name

Type' ___~____.

1 :

z

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 23 30 31 32 33 34 :z 37 :: 40 41 42 43 44 45 46 47 48 49 50 ::

._.----_

(20s)-loo-diacholest-13(17)-ene (2~)-lOa(diacholest_13(17)-ene (Z(fR)-lOP(H)-diacholest-13(17)-ene (2&Q)-lOa(diacholest-13(17)-ene (2fX)-24-nethyl-10a(H)-dL%choleat-13(l7)-ene (2QQ)-24-methyl-LOa(H)-dincholest-13(17)-ene (2U )-24-ethyl-lOa(diacholest-13(17)-ene 120R~-24-ethvl-100fHl-diacholest-l3fl7~-ene ~2(Ylj-24-ethyl-l0o~Hj-diacholest-l3~l7j-~~~ Partially rearranged cholestene Partially rearranged 24-ethylcholestene Partially rearranged 24-ethylcholestene (2W)-S=(H)-cholest-14-ene (20s)-50(H)-cholest-8(14~-ene ~2Wj-5a~Hj-cholest-l4-ene (Z&Q)-Sa(H)-cholsst-8(14)-ene (20s)-24-methyl-5a(H)-cholest-14-ene (2~)-24-inethyl-5atX)-chalest-8(14)-ene (2~)-24-nethyl-S~(H)-chalest-l4-e~e (2~)-24-methyl-5~(H)-chole~t-8(14)-ene (20s)-24-ethyl-5a(H)-cholest-l4-ene (Zas)-24-ethyl-5a(H)-cholest-E(14)-ene (2O.Q-24-ethyl-5a(H]-cholest-l4-ene (20R)-24-ethyl-5a(H)-cholest-8(14)-ene (2QQ)-rearranged-1OP(H)-spirocholest-l3(l7)-ene (ZQR)-rearranged-lOaO_spitocholest-13(17)-ene (2Cb")-rearranged-1O~(W)-spirocholest-l3(l7)-er1e (2Rs)-rearranged-l0~(H]-~pirocholest-13(l7)-er1e (2&Q)-5P(H)-spirocholest-l3(17)-ene (2&Y)-SP(H)-spirochol.est-13(17)-ene (2W)-SO(H)-spirocholest-13(17)-ene (20s)-Sa(H)-spirocholest-13(17)-ene (2CA)-24-methyl-5j3(H)-spirocholest-l3(l7)-ene (20S)-24-methyl-5~(H)-spirocholest-l3(17)-er1e (2oR)-24-methyl-5a(H)-spirocholest-l3(l7)-ew (2QY)-24-methyl-5%(H)-spirocholest-13(17)-ene (2W)-4a,24-dimethyl-5a(H)-spicocholest-l3(17)-ene (2~)-4a,24-dimethyl-5a(H)-spirocholest-13(17)-ene (2~)-4~,24-dimethyl-5a(X)-spirocholsst-~3(~7)-ene (2~)-4P,24-dimethyl-Sa(H)-spirocho~~st-l3(17)-ene (2aR)-24-ethyl-Sf3(X)-spirocholest-l3(17)-ene (2Or)-24-ethyl-S@(H)-spirocholest-13(17)-en@ (2aP)-24-ethyl-5a(H)-spirocholest-l3(17)-ene (2aS)-24-ethyl-5=(H)-spirocholest-13(17)-ene Cholest-4-ene k(H)-cholest-Z-ene Cholest-5-ene Ring C monoaromatic steroid (2oR)-5a(H),l4a(H),l?a(H)-cholestane (2ClY)-Sa(H),14a(H),l7~(H)-cholestane (2QQ)-5a(H),14!3(H),17P(H)-cholastane (2aC)-Sa(H),14P(H),17P(W)-cholestane

II IS II II IP 11 II II ix III 1x1 III

1 I I I I I I I I I r i c V V ” IV IV IV I” IV IV IV IV IV IV I” IV IV IV I” I” I I I I I I

-_*TYPE

refers

to

general structures in Figure 1.

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Occurrence of sterenes and steranes in sediment 80

ENSMINGERet al., 1978), although studies concerning their diagenesis are limited (e.g., WOLFF et al., 1986).

B

fl

60 EXPERIMENTAL The sediment sample used was a marl from a Messinian (late Miocene) evaporitic basin located in the northern Apeninnes, Italy (see TEN HAVEN et al., 1985, for a detailed sample description). The powdered sample (300 g) was soxhlet extracted with dichloromethane/methanol (9:l; 24 h). Elemental sulphur was removed over activated copper turnings. AFter removal of the solvent the extract was chromatographed on silica (25 g, 0.030-0.075 mm) and the hydrocarbon fraction obtained by elution with hexane ( 1.5 column volumes). This fraction was analysed by GC and GC/MS. It was also further fractionated by HPLC (c& WOLFF et al., 1985). Thus, an aliquot in dichloromethane (25 ~1) was injected onto the column (Soherisorb 5W 0DS2: 250 X 10 mm id.: Phase Sep. Ltd.). Acetone/ water (96:4; v/v) was the mobile phase (4 ml mik’) and fractions (l-10) were collected every 3 min. The solvent was removed (toluene azeotrope) and each fraction was analysed by GC and, in some cases, GCIMS. Rearrangement products from C2,, Cz8 and Cz9 A5 sterenes were available from previous studies (PEAKMAN and MAXWELL,1988a; PEAKMANet al., 1988) as were those from Sa(H)-cholest-7-ene (C2,; PEAKMAN and MAXWELL, 1988b). Rearrangement products from 24-methyl-5a(H)-cholest-7-ene (C,,) were obtained in a manner analogous to that for their C2, homologues (PEAKMANand MAXWELL, 1988b). The rearrangement/hydrogenation experiments on 5a(H)-cholest7-ene were carried out by stirring the sterene (25 mg) in glacial acetic acid (125 ml) and perchloric acid (12.5 ml) at ambient temperature and pressure. Samples (25 ml) were withdrawn at intervals and hydrogenated in the same medium over 10% platinum on charcoal for 16 h. The products were extracted with dichloromethane (3 X 10 ml) and the combined extracts neutralised (saturated sodium bicarbonate) and dried (magnesium sulphate). After filtering and removal of the solvent under reduced pressure, the crude material was chromatographed on alumina (1 g). Elution with hexane afforded the hydrocarbon products. GC analyses were performed on a Carlo Erba 5300 gas chromatograph (on-column injection) fitted with a 25 m X 0.3 mm fused silica capillary column coated with OV- 1 (df = 0.17 pm). Helium was the carrier gas. The sample was injected in hexane at 40°C and then moerammed to 100°C at 10°C min-’ and then to 300°C at 4°C mic’. GC/MS was performed on a similar chromatograph (on-column injection) fitted with either a 25 m X 0.3 mm fused silica capillary column coated with OV- 1 (df = 0.17 ,um) or a 30 m X 0.3 mm fused silica capillary column coated with DB- 17 (df = 0.17 pm). Helium

4 X6

’

3

A

Retention

Time -

FIG. 2. Partial m/z 257 chromatograms (DB- 17 stationary uhase) of the marl aliphatic hydrocarbon fraction showing the distributions of Alx”) diasterenes (l-7,9) and the presence of a partially rearranged sterene (10) and Czs-Czs A*(14)and At4 Sa(H)-sterenes (19-24).

t

40

40

20

20

0

C27 t28

C29

0

C27 C28 C29

C 60

C27 C28 C29

0

(27

C28 C29

FIG. 3. Histograms of carbon number distributions and extents of C-20 isomer&ion for: (a) A’3(‘7)lOa(H)diasterenes; (b) 5a(H),14a(H),l7or(H)-steranes; (c) 5o1(H),14fl(H),17fl(H)-steranes and (d) Alx”) Sa(H)-spirosterenes in the marl aliphatic hydrocarbons. (2OR)isomers are shaded in black and the (20S)Gsomers hatched.

was the carrier gas. The gas chromatograph was interfaced directly with a Finnigan 4000 mass spectrometer (quadrupole; ionising voltage 35 eV, filament 350 PA) with a source temperature of 25O’C. The spectrometer was scanned cyclically (1 s) from m/z 50 to 550, or was operated in the multiple ion detection mode. Data were acquired and processed using a Finnigan INCOS data system. The GC conditions were similar to those described above except when co-injections with standards were performed; in this case the oven was programmed from lOO-300°C at 1“C min-‘. Analyses of carbon number distributions were made by GC/MS using peak areas From the appropriate mass chromatograms (+S%). RESULTS Steroid alkenes thought to be derived from A5 sterols The distribution of AL3(“) lOa(diasterenes in the hydrocarbon fraction of the marl is illustrated by the m/z 257 chromatogram in Fig. 2. It is dominated by the C27 components (2, 4) with the Cz7, Czs (5, 6) and Cz9 (7, 9) relative abundance being ca. 82%, 7% and 1 l%, respectively (based on peak areas in the mfz 257 chromatogram; Fig. 3a). The complete separation ofthe Czs (20R)-A’3(‘7) lOa(diasterene (6) from the CZ9 (2OS)-A ‘3(‘7) lOLu(H)-diasterene (7) on DB17 allowed measurement of the extent of C-20 isomerisation for each carbon number (all ea. 60:40 in favour of the (2OR) isomer). Minor components of the backbone rearrangement of cholest-Gene and 5-ene (C,,), namely the A’3(‘7) IO@(H)diasterenes (1, 3) and a partially rearranged sterene (10; cf. PEAKMAN and MAXWELL, 1988a) are present (Fig. 2; confirmed by co-injection with authentic compounds on OV-1 and DB- 17) whereas the higher homologues are virtually absent. In the m/z 206 chromatogram (Fig. 4a) the CZ7 rearranged spirosterenes (25-28) can be seen (cJ, PEAKMAN and MAXWELL, 1988a), although the higher homologues were not readily observed in the m/z 220 and 234 mass chromatograms. Some of the minor C&s and CZ9 products (e.g.,

2004

1’. M. Peakman t’r (I(.

Retention

Time ---t

-z m/z

z + c -

respectively; Fig. 3d). The extents of isomerisation at C-20 (R):(S) for the homologues are all basically the same (ca 52: 48) being slightly more advanced than that
16

206

m/z 234 3 61

i

Retention

i’ A_iiu

42

Time

-

FIG. 4. Partial m/z 206, 220 and 234 chromatograms of the marl aliphatic hydrocarbon fraction showing the distributions of A’3(‘7) spirosterenes: (a) OV- 1 stationary phase and (b) DB- 17 stationary phase.

Retention

Time

-

AL3(“) lO~(H)-diasterene (8) and partially rearranged sterenes (11 and 12)) were, however, detected in some of the HPLC fractions (e.g., Fig. 5a) and their presence confirmed by coinjection with authentic compounds on the two phases. Cholest-4-ene (45), 5a-cholest-2-ene (46) and cholest-5ene (47) were detected as minor components of HPLC fraction 7 (Fig. 5a). Higher homologues were not observed. Steroid alkenes thought to be derivedfrom (and A88(14)) sterols

23

A7

The distribution of A’3(‘7)spirosterenes, exemplified by the m/z 206, 220 and 234 chromatograms, is illustrated in Fig. 4. The major A”(“) So!(H)-spirosterenes (31, 32, 35, 36,43, 44) are clearly present, as are minor amounts of the proposed SD(H)-isomers (TEN HAVEN et al., 1985; 29, 30, 33, 34, 41, 42). The relative abundance of Cz7, Cz8 and Cz9 A’3(‘7)5c~(H)spirosterenes are ca. 24, 33 and 43%, respectively (based on peak areas in the mJz 206, 220 and 234 chromatograms,

Retention

Time

-

FIG. 5. Partial m/z 2 15 and 257 chromatograms (DE 17 stationary phase) showing the distributions of A8(14) and Al4 Sa(H)-sterenes in two HPLC fractions of the marl aliphatic hydrocarbons: (a) fraction 7 and (b) fraction 8.

2005

Occurrence of sterenes and steranes in sediment

standards were carried out on these fractions on both phases. It should be noted that on OV-1 (not shown) the Asu4)5a(H)sterenes co-elute with their Ai4 Sa(H)-counterparts (cf, PEAKMAN and MAXWELL, 1988b). Careful analysis of the HPLC fractions indicates that further laboratory rearrangement products of the A’3(‘7)SLY(H)spirosterenes (e.g., A ‘7(20)14/3-methyl- 1g-nor- 13/3(H)-components; PEAKMANand MAXWELL, 1988b) do not occur in the marl above the detection level.

to those of their 4desmethyl counterparts (cu. 52:48). The m/z 257 chromatogram (Fig. 2) indicates the presence, in low abundance, of sterenes with the retention times of C2&z9 (20R)- and (2OS)-Aso4) and Al4 Sa(H)-components (19-24) as confirmed by co-injection of the Cz8 standards on the two phases (OV-1 and DB-17). Both the A8(14)and Al4 %x(H)-sterenes are also isomeric at C-20 (Figs. 2 and 5; 1324), although their low abundances prevented accurate measurements of the (20R):(20S) ratios. Their carbon number distributions, however, are in accord with those of the A’3(‘7’ Sa(H)-spirosterenes (i.e., Cz9 > Cz8 > C2,). The abundances of the Cz7 (13-16) and (20s)~Cz8 (17, 18) homologues were difficult to determine due to their presence in lower abundance and co-elution with other sterenes. CC/MS analysis (DE 17) of HPLC fractions 7 and 8 (Fig. 5) allowed, however, spectra of the sedimentary components to be obtained (Fig. 6) and co-injection experiments with both the Cz, and Cz8

are also comparable

251

loo-

E

Components arising from reduction of steroid alkenes The distributions of (~OR)-~U(H),~~CX(H),~~~(H)- and (2OR)- and (20S)-5a(H),l4P(H),17/3(H)-steranes (based on peak areas in the m/z 217 and 2 18 chromatograms, respectively) are illustrated in Figs. 3b and c, respectively (c_f:TEN HAVENet al., 1985). The relative abundances of C2’, cz8 and C2, (20R)-5a(H),14~u(H),17~u(H)-steranes are cu. 75, 8 and

loo

94

100

I~"'~~~~

200

300

I;-

400

100

200

300

400

FIG. 6. Mass spectra of selected sedimentary Asd4’and Al4 Sa(H)-sterenes and synthetic standards for comparison as analysed by CC/MS on DE1 7 stationary phase: (a) synthetic 24-methyl-k(H)-cholest-14-ene (19); (b) synthetic SdH)-cholest-8( 14bne (16); (c) sedimentary Sa(H)-cholest-14-ene (15)-the spectrum indicates the presence of cholest4ene (45) as seen by ions at m/z 108,215,355 and 370 and Sa(H)-cholest-2-ene (46) as seen by ions at m/z 203,215, 301,316, 355 and 370; (d) sedimentary Sa(H)-cholest-8( 14)-ene (16)-the spectrum indicates the presence of cholest5-ene (47) as seen by ions at m/z 275 and 301; (e) sedimentary 24-ethyl-Sa(H)-cholest-14-ene (23); and (f) sedimentary 24-ethyl-5o(H)-cholest-8( 14)-ene (24). For reference purposes the reader is referred to FWILP (1985) for related sterene mass spectra.

2006

T. M. Peakman et ul.

(2W)sa(H),14Q(H),17a(H) -_.___

(2M)5u(H),14P(H),17P(H)

(2W)WH),14P(H),17!3(H)

Cz7

1.000

0.043

0.042

ClS

0.106

0.060

0.055

C..

0.231

0.072

0.069

*Nommlised to (2OR)-Sa(H),14a(H),17a(H)-cholestane (49,. the measurements have been made from peak areas in themjz 217 chromatogram for the (2~)-5a(H),14a(H),17a(H)-steranas and from peak areas in the m/z 218 chromatogram for the (2~)-end (20s )-5a(H),14P(H),17P(H)-steranes.

17% and those for the &z(H), 14/3(H),17/3(H)-steranes ca. 25, 33 and 42%, respectively. Comparisons between the two series are given in Table 2. The (20R):(20S) ratios for the Sa(H), 14/3(H)-sterane homologues are all basically the same (ca. 51:49). Only trace amounts of (20S)-5a(H),14a(H),17cu(H)-steranes were observed. Series of 4a- and 4@-methyl-Sa(H), 14a(H), 17a(H)-steranes (C&C& are present, but the tentatively proposed 4a- and 4&methyl-&x(H), 148(H), 17@(H)-steranes show only Cz9 components in significant relative abundance (TEN HAVEN IIJ~ al., 1985). Diasteranes and 4-methyldiasteranes were not detected. Rearrangement/hydrogenation on Sa(H)-cholest- 7-ene

experiments

In order to gain further evidence for the proposed intermediacy of Sa(H), 14&H), 17/3(H)-steranes, Sa(H)-cholest-7ene was stirred in an acidic medium at ambient temperature and pressure. The product distribution obtained after 175 h and subsequent hydrogenation (Fig. 7) shows the major sterane components to be (2OR)- and (20s)~Sol(H),14B(H),17/3(H)-cholestanes (51,52; 77% of the sterane components) with a (20R):(20S) ratio of 59:41. (2OR)- and (20s) 5a(H), 14ru(H), 17a(H)-cholestanes are also present (49, 50; 23% of the sterane components) with a (2OR):(2OS) ratio of 65:35. A component arising from disproportionation is also present (48); it appears from its mass spectrum (weak molecular ion at m/z 366, base peak at m/z 253), to be aroma&d in ring C. Interestingly, (20R)-5a(H),14P(H), 17a(H)-cholestane, which is also found in sediments, was observed in low abundance after short periods of acid treatment and subsequent hydrogenation but was subsequently not detected. The other major features, resulting from short periods of acid treatment and subsequent hydrogenation, were the lower amounts of (20s)-5a(H),14cz(H),l7~(H)and (20S)-5a(H),14P(H),17fi(H)-cholestanes (52, 50) relative to their (20R)-isomers.

isomer&ion at C-20 in the A‘3”7) 1Oa(H)-diasterenes mdicates that the backbone rearrangement is well advanced in the marl (I$, BRASSELLet al., 1984; PEAKMAN and MAXWELL, 1988a). The almost identical ratios for C-20 isomerisation within the carbon number homologues of the latter is in contrast to the observation of CURIALE (1987), who reported different extents for the Kishenehn formation of northwest Montana (U.S.A.), although his measurements were hampered by co-elution of the C2, (20R)-A’7”7’ 1Ocu(H)diasterene (6) with the Cl9 (20S)-A’3(‘7) lOa(diasterene (7) on the column used (cf BRASSELLet al., 1984). The relative abundance of Cl,, Cz8 and Cz9 ““7) lOa( diasterenes (Fig. 3a) suggests inputs of A5 sterols with similar carbon number distributions into the original surface sediment since the lOa(diasterenes arise from A5 sterenes (RUBINSTEINet al., 1975; PEAKMANand MAXWELL, 1988a) which in turn arise from As sterols via A3,s steradienes (DE LEEUW et al., 1989). The presence of Scr(H)-cholest-2-ene, co-occurring with cholest-4-ene, cholest-5-ene (46, 45 and 47, respectively; Fig. 5a) and abundant A’j”” diasterenes, suggests that there is indeed no isomerisation of the A’ bond to A4 at this level of maturity (~5, DE LEEUW t’I&, 1989). The absence of diasteranes and 4-methyldiasteranes in the marl is in keeping with its immaturity. Components arising_fkom A7 (and Aaci4))stero1.c The presence of Asti4) and Ai SLu(H)-sterenes in the marl indicates inputs of A7 (and A8(‘4))Sa(H)-sterols into the original sediment, since in the laboratory a A5 double bond does not isomerise to these positions (PEAKMAN efal., 1988). Their occurrence also provides the first geochemical link between A7 (and Aa(14))Sa(H)-sterols and the At3(17)Scu(H)-spirosterenes which have been found in many sediment samples (PEAKMAN etal., 1984; BRASSELLet al., 1984; TEN HAVEN et al., 1985, 1989; CURIALE, 1987). The presence of A’3(‘7) S@(H)-spirosterenes likewise indicates an origin from A’ SB(H)-sterols, which could also arise in part from reduction of the A5 double bond of A5x7sterols which have been tentatively identified in sediments (GAGOSIAN and FARRINGTON,

DISCUSSION

The present detailed study confirms and extends aspects of earlier studies of the low temperature diagenesis of sterenes (see Introduction). Components arising from As sterols The minor amounts of cholest-4-ene (45) and cholest-5ene (47) in HPLC fraction 7 (Fig. 5) and the high degree of

Retention

Time

-

FIG. 7. Reconstructed total ion chromatcgram (OV-1 stationary phase) of products obtained from rearrangement/hydrogenation of Sa(H)-cholest-7ene.

Occurrence of sterenes and steranes in sediment

2007

accompanied by a minor amount of the 5a(H),14P(H),17cY(H)isomers.This indicates that a minor amount of (20X)5a(H),14a(H),l?a(H)and (20R)- and (20S)-5a(H),14D(H),lIla(H)-steranes can be formed in immature sediments via this pathway. The formation of the 5a(H),1413(H),17~(H~ste~es is explicable in terms of rearrangement of the A’Yi7) 5~(H~spirosterenes to transient A” 5a(H), 17/3(H)-sterenes which are then reduced (cJ, CASPI et al., 1975; PEAKMAN and MAXWELL, 1988b). These rearrangement/reduction pathways are summarised in Fig. 8 in terms of the diagenesis of A7 sterols. Additional evidence for the conversion of A’y’7) 5a(H)spirosterenes to 5a(H), 14@(H),17&H)-steranes in immature sediments comes from comparison of carbon number distributions. The dist~butions of the S~(H~,l4~(H),l7~(H~ steranes (Fig. 3cf and the 5ti(H), 140!(H),17cu(H)-steranes (Fig. 3b) in the marl are different, whilst the Scu(H),14@(H),17/J(H)sterane distribution closely matches that of the A’3(17)5a(H)spirosterenes (Fig. 3d). The (2OR):(2OS) ratios of the Ar3(17) Sa(H)-spirosterenes and 5a(H), 14fl(H),17/3(H)-steranes are

1978). The presence of (20S)-A804’ and (2OS)-Al4 %X(H)sterenes is expected from laboratory studies on the rearrangement of A7 k(H)-sterenes (PEAKMAN and MAXWELL, 1988b). The absence of further rearrangement products of the AhlX”) Scr(H)-spirosterenes (e.g., A’700) 14@-methyi- 18nor- 13~(~~sterenes; PEAKMAN and MAXWELL, 1988b) in this sample suggests that rearrangement of the double bond out of the steroid nucleus is less favourable than its reduction. Preliminary laboratory studies (PEAKMAN and MAXWELL, 1988a,b) have indicated the formation of 5a(H),14@(H),17fi(H)-steranes from hydrogenation of A13(‘7)Sa(H)-spirosterenes in acidic media. This is supported by the laboratory rearrangement/hydrogenation experiments on k(H)-cholest7-ene. Thus, A’ Sa(H>sterenes are rearranged into A8u4fand Al4 5~tH)-sterenes and then to A’y’7) 5~H~spir~terenes which undergo isomer&&ion at C-20. This rearrangement is reversible, and so not only are the Aso4) and Ai4 5ar(H)-sterenes regenerated but their (20X)-isomers are also formed (c$., PEAKMANand MAXWELL, 1988b). Reduction of the C20 isomeric Al4 sterenes gives Srw(H),14a(H), 17a(H)-steranes

BlOLOGfCAL CONTRIBUTION

I

4420

I

I

-HZ0

k.\

-Hz0

H+

(+ trace

14~(H)?)

PrG. 8. Proposed diagenesis of A’ (and A‘(r4)sterols) (taken in part from PEAKMAN and MAXWELL, 1988a; and DE The stereochemistry is not defined at C-5 since the scheme operates for both Scu(H)and S@(H)

LEEUW et ai., 1989).

configurations.

2008

T. M. Peakman et al.

also identical (within experimental error). This indicates that isomerisation at the chiral centres of the steranes is not occurring directly at this level of maturity. It is likely, therefore, that the SLY(H),14/?(H), 17/3(H)-steranes in immature sediments arise from A’ (and As(14))%X(H)sterols via (A’*‘), A2,8(‘4)and A2,14Sa(H)-steradienes, (A’). A8(14)and Al4 %x(H)-sterenes, A IX”) Sa(H)-spirosterenes and Al4 SLY(H),17/3(H)-sterenes (Fig. 8). This situation can occur in hypersaline environments where a larger contribution of the necessary precursor sterols, relative to A5 sterols and Sa(H)-stanols, is often encountered (e.g., BOON et al., 1983; DE LEEUW et al., 1985). It should be pointed out, however, that A’ (and A8(14))Sa(H)-sterols are also abundant in other environments (e.g., CRANWELL, 1982; SMITH et al., 1982, 1986; ROBINSON et al., 1984, 1986; VOLKMAN, 1986). Thus, the presence of A’ sterols in Lake Kinneret has been suggested as indicating an input from Chlorophyta plankton (ROBINSON et al.. 1986). It is possible, therefore, that “anomalous” relative

amounts of 5a(H), 14fl(H),l7/3(H)-steranes in immature sediments need not to be confined to hypersaline environments. The distributions of 4-methylsteroid hydrocarbons in the marl are in agreement with the above conclusions. Thus, the major Ai3(“j 4-methyl-Sa(H)-spirosterenes and 4-methyl5&(H), 14P(H),l7/3(H)-steranes are the Cz9 homologues. They also occur with similar ratios of isomers at C-4 and C-20. These C29 4-methyl-steroid hydrocarbons are probably derived, therefore, from A’ and/or A8(‘4)4-methylsterols. To what extent the formation of 5a(H),l4P(H), 17P(H)steranes in immature sediments reflects the sterane distributions in more mature sediments and crude oils remains to be seen. Since absolute sterane abundances are known to decrease with increasing maturity (RULLK~TTER et al., 1984), it is possible that the observed increase in relative abundance of the 5a(H),l4&H),l7@(H)-steranes may also be due to preferential destruction of the thermodynamically less stable 5a(H), 14a(H), 1‘la(H)-isomers in addition to the traditional isomerisation pathway. These studies confirm previous warnings (MACKENZIE, 1984; TEN HAVEN et al., 1986) concerning the use of 5a(H), 14a(H), 17a(H)- to 5a(H), 14/3(H),17@(H)-sterane ratios in maturity assessment. It should also be stressed that initial minor quantities of (20S)-5a(H), 14a(H), l’la(H)-steranes can also be formed in immature sediments via sterene intermediates such as the (20s) Al4 Sa(H)-sterenes (13, 17, 21) identified herein, although this does not appear to seriously affect the use of the (20R):(20S) isomerisation ratio in the 5a(H),l4~~(H),l7a(H)-steranes (e.g., RULLK~TTER and MARZI, 1988). This anomaly

in the use of 5a(H),l4a(H), 17a(H)- to 5a(H), 14@(H),17/3(H)-sterane ratios in maturity assessment would appear to be recognisable from comparison of carbon number distributions between the two sterane classes and from low amounts of (20S)-5a(H), 14a(H), 17a(H)-steranes (e.g., Fig. 3b and c, and Table 2).

CONCLUSIONS 1. Seriesof (2OR> and (20S)-A8(‘4)and Al4 Scz(H>sterenes have been identified in a hypcrsaline deposit, from an immature northern Apennines marl, where they co-occur with A’3(‘7)Sa(H)-spirosterenes and transient (20R)- and (2OS)-

Al4 5o1(H),l7/3(H)-sterenes. It is likely that these isomeric sterenes arise from A’ (and A8(14)) Sa(H)-sterols viu (A’,‘), A2,8(14)

and

A&l4

Sol(H)-steradienes. 2. Reduction processes, via the Ai4 5c~(H),17&H)-sterenes, can give rise to the 5@(H),14/~‘(H),17/3(H)-steranes present in this sample in anomalously high relative abundances. This pathway is supported by laboratory rearrangement/hydrogenation experiments on Scr(H)-cholest-7-ene. ‘These experiments also indicate that minor amounts of (2OS)5a(H), 14~u(H),17cY(H)-steranes can be formed by this route. 3. Comparison of carbon number distributions and (20R): (20s) ratios indicates that the 5a(H), 14/3(H),17@(H)-steranes are indeed related to the A”(“) Scu(H)-spirosterenes and As(‘4) and At4 Sa(H)-sterenes and not to the 5~y(H),14cu(H),17a(H)steranes. This is also apparent for 4-methyl-Sa(H),14/3(H),17@(H)-steranes and A““” 4-methyl-5
REFERENCES ABBOTTG. D., LEWISC. A. and MAXWELLJ. R. (1985) The kinetics of specific organic reactions in the zone of catagenesis. Phil. Trans. Roy. Sot. London A 315, 107-122. AKPORIAYED. E., FARRANTR. D. and KIRK D. N. ( 1981) Deuterium incorporation in the backbone rearrangement of chotest-5-ene. J. Chem. Res. (S), 2 10-2 1 I. BEAUMONTC., BOUTILIERR., MACKENZIEA. S. and RULLK~TTER J. (1985) Isomerization and aromatization of hydrocarbons and the paleothermometry and burial history of Alberta Foreland Basin. Amer. Assoc. Petrol. Geol. Bull. 69, 546-566. L@ONJ. J., HINES H., BURLINCAMEA. L., KLOK J., RLJPSTRA W. I. C., DE LEEUWJ. W., EDMUNDSK. E. and EGLINTONG. (1983) Organic geochemical studies of Solar Lake laminated cyanobacterial mats. In Advances in Organic Geochemistry 1981 (eds. M. BJ~ROY et a/.), pp. 207-227. J. Wiley & Sons, Chichester. BRASSELLS. C., MCEVOYJ., HO-ANN C. F., LAMBN. A., PEAKMANT. M. and MAXWELLJ. R. (1984) Isomerisation, rearraneement and aromatisation of steroids in distinguishing early stages of diagenesis. In Advances in Organic Geochemistry 1983 (Eds. P. A. SCHENCK,J. W. DE LEEUWand G. W. M. LLJMBACH); Org. Geochem. 6, pp. 1l-23. CASPI E., DUAX W. L., GRIFF’INJ. F., MOREAUJ. P. and WITSTRUCKT. A. (1975) An unusual backbone rearrangement. The formation of 5a, 17c+cholest- 14-en-38-01 acetate from 5or-cholest8(14)-en-38-01 acetate. J. Org. Chem. 40, 2005-2006. CRANWELLP. A. (1982) Lipids of aquatic sediments and sedimentary particulates. Prog. Lipid Res. 21,27 l-308.

Occurrence of sterenes and steranes in sediment CURIALE J. (1987) Steroidal hyphens

of the Kishenehn formation northwest Montana. Org. Geochem. 11,233~244. ENSMINGERA., JOLY G. and ALBRECHTP. (1978) Rearranged steranes in sediments and crnde oils. TetrahedronLett., 1575-1578. FRIED J. and EDWARDSJ. A. (1972) Organic Reactions in Steroid Chemistry. Vol. I. Van Nostrand Reinhold, New York. GAGOSIANR. B. and FARRINGTONJ. W. (1978) Sterenes in surface sediments from the southwest African shelf and slope. Geochim Cosmochim. Acta 42, 1091-l 101. VAN GRAAS G., BAASJ. M. A., VANDE GRAAF B. and DE LEEUW J. W. (1982) Theoretical organic geochemistry. I. The thermodynamic stability of several cholestane isomers as calculated by molecular mechanics. Geochim. Cosmochim. Acta 46,2399-2402. TEN HAVEN H. L., DE LEEUW J. W. and SCHENCKP. A. (1985) Organic geochemical studies of a Messinian evaporitic basin, northern Apennines (Italy). I: Hy~~~~n biological markers for a hypersaline environment. Geochim. Cosmochim.Acta49,21812191. TEN HAVENH. L., DE LEEUWJ. W., PEAKMANT. M. and MAXWELL J. R. (1986) Anomalies in steroid and hopanoid maturity indices. Geochim. Cosmochim. Acta 50,853-855. TEN HAVEN H. L., DE LEEUW J. W., SINNINGHEDAMSTBJ. S., SCHENCKP. A., PALMERS. E. and ZUMBERGEJ. E. (1988) Ap plication of biological markers in the recognition of palaeohypersaline environments. In Lacustrine PetroleumSource Rocks (eds. A. J. FLEET, K. KELTS and M. R. TALBOT);GeologicalSociety Special PublicationNo. 40. pp. 123-I 30. TEN HAVENH. L., RULLK~TTERJ. and WELTED. H. (1989)Steroid biological marker hydrocarbons as indicators of organic matter diagenesis in deep sea sediments: Geochemical reactions and inBuence of different heat flow regimes. GeoZ.Rund. (in press). KIRK D. N. and SHAW P. M. (1975) Backbone rearrangement of steroidal 5-enes. J. Chem. Sot.: Perkin Trans. I. 2284-2294. DE LEEW J. W. and BAASM. (1986) Early-stage diagenesis ofsteroids. In Methodsin Geochemistryand Geophysics24. BiologicalMarkers in the Sedimentary Record. (ed. R. B. JOHNS), pp. 101-123. Elsevier, Amsterdam. DE LEEUW J. W., SINNINGHEDAMSTE.J. S., KLOK J., SCHENCK P. A. and BOON J. J. (1985) Biogeochemistry of Gavish Sabkha sediments 1. Studies on neutral reducing sugars and lipid moieties by gas chromatography-mass spectrometry. In EcologicalStudies, Vol. 53: Hypersaline Ecosystems (eds. G. M. FRIEDMANand W. E. KRUMB~IN),pp. 350-367. Springer-Verlag, Berlin. DE LEEUWJ. W., Cox H. C., VAN GRAASG.. VANDE MEER F. W.. PEAMAN T. M., BAASJ. M. A. and VANDE GRAAF B. (1989) Limited double bond i~me~~tion and selective hy~ogenation of sterenes during early diagenesis. Ge~him. Cosmochim. Acta 53,903-909. MACKENZIEA. S. (1984) Application of biological markers in petroleum geochemistry. In Advances in PetroleumGeochemistry(eds. J. BROOKS and D. H. WELTE), Vol. 1, pp. 115-2 14. Academic Press, London. MACKENZIEA. S. and MCKENZIED. (1983) Isomerization and aromatization of hydrocarbons in sedimentary basins formed by extension. Geol. Mag. 120,4 1l-470. MACKENZIEA. S., PATIENCER. L., MAXWELLJ. R., VANDENBROUCKEM. and DURAND B. (1980) Molecular parameters of maturation in the Toarcian Shales, Paris Basin, France-I. Changes in the configuration of acyclic isoprenoid alkanes, steranes and triterpanes. Geochim. Cosm~him. Acta 44,1709- 172 1. MACKENZIEA. S., BRA.%ELLS. C., EGLINTONG. and MAXU’ELL J. R. (1982) Chemical fossils: The geological fate of steroids. Science 271,491-504. PEAKMAN T. M. and MAXWELLJ. R. (1988a) Acid-catalvsed rearrangements of steroid alkenes-I. Rearrangement of Sk-cholest‘I-ene. J. Chem. Sot.; Perkin Trans. I. 1065-1070. PEAKMANT. M. and MAXWELLJ. R. (1988b) Early diagenetic pathways of steroid alkenes. In Advancesin OrganicGeochemistry1987 (eds. L. MATTAVELLIand L. NOVELLI);Org. Geochem. 13,583592. PEAKMAN 'I'. M., LAMBN. A. and MAXWELLJ. R. (1984) Naturally occurring Spiro steroid hydrocarbons. TetrahedronLett. 25,349352. PEAKMANT. M., ELLJSK. and MAXWELLJ. R. (1988) Acid-catalysed rearrangements of steroid alkenes-II. A re-investigation of the

2009

backbone r~~~~ent of cholest-5-ene. J. Chem. Sot.; Perkin Trans. I, 1071-1075. PETROVA. A., P~JSTIL’NIKOVA S. D., ABRI~JNTINA N. N. and KAGRAMONOVAG. R. (1976) Petroleum steranes and triterpanes. Neftkhimiya 16,41 l-427 (in Russian). PHILP R. P. (1985) Methods in Geochemistryand Geophysics23. Fossil Fuel Biomarkers. Applicationsand Spectra. Elsevier, Amsterdam. ROBINSONN. A., CRANWELLP. A., FINLAYB. J. and EGLINTONG. (1984) Lipids of aquatic organisms as potential contributors to lacustrine sediments. In Advances in Organic Geochemistry1983 (eds. P. A. SCHENCK,J. W. DE LEEUWand G. W. M. LIJMBACH); Org. Geochem. 6, 143-152. ROBINSONN. A., CRANWELLP. A., EGLINTONG., BRASSELLS. C., SHARPC. L., GOPHENM. and POLL~NGHER U. (1986) Lipid geochemistry of Lake Kinneret. In Advancesin OrganicGeochemistry 1985 (eds. D. LEYTHAEUSER and J. RULLK&TER); Org. Geochem. 10,733-742. RUBINSTEINI. and ALBRECHTP. (1975) The occurrence of nuclear methvlated steranes in a shale. J. Chem. Sot.; Chem. Commun., 957-658. RUBINSTEINI., SIESKIND0. and ALBRECHTP. (1975) Rearranged sterenes in a shale: Occurrence and simulated formation. J. Chem. Sot.; Perkin Trans. I, 1833-l 836. RULLKOTTERJ. and MARZI R. (1988) Natural and artificial maturation of biological markers in a Toarcian shale from northern Germany, In Advances in Organic Geochemistry I987 (eds. L. MATTAVELLIand L. NOVELLI);Org. Geochem. 13,639-64.5. RULLKOT~ERJ., MACKENZIEA. S., WELTE D. H., LEYTHAEUSER D. and RADKE.M. (1984) Quanti~tive gas chromato~phy-rna~ spectrometry analysis of geological samples. In Advancesin Organic Geochemistry 1983 (eds. P. A. SCHENCK,J. W. DE LEEUWand G. W. M. LIJMBACH);Org. Geochem. 6,817-827. RULLK~TTERJ.. SPIRO B. and NISSENBAUMA. (1985) Biological marker characteristics of oils and asphalts from carbonate source rocks in a rapidly subsiding graben, Dead Sea, Israel. Geochim. Cosmochim. Acta 49, 1357-l 370. RULLK~TTERJ., MEYERSP. A., SCHAEFERR. G. and DUNHAM K. W. (1986) Oil generation in the Michiaan Basin: A biological marker and carbon isotope approach. In Advances in Organic Geochemistrv1985 leds. D. LEYTHAEUSER and J. RULLK~TTER); Org. Geochem. 10, 359-375. SEIFERTW. K. ( 1980) The impact of Treibs’ discovery of porphyrins on present day biological marker organic geochemistry. In Proceedings of the Treibs’ Internutional Sym~sium (ed. A. A. P~HNOWSKY), pp. 13-35. H~b~~~k, Wombs. SEIFERTW. K. and MOLDOWANJ. M. (1979) The effect of biodegradation on steranes and terpanes in crude oils. Geochim. Cosmochim. Acta 43, 11- 126. SEIFERTW. K. and MOLDOWANJ. M. (1981) Paleoreconstrnction by biological markers. Geochim. Cosmochim. Acta 45,783-794. SINNINGHEDAMST~J. S., TEN HAVENH. L., DE LEEWWJ. W. and SCHENCK P. A. ( 1986) Organic geochemical studies of a Messinian evaporitic basin, northern Apennines (Italy). II. Isoprenoid and nalkyl thiophenes and thiolanes. In Advancesin OrganicGeochemistrv I985 (ads. D. LEYTHAEUSERand J. RULLK~TTER);Org. Ge&hem. lb, 791-805. SMITH D. J., EGLINTONG., MORRIS R. J. and POUTANENE. L. ( 1982) Aspects of tbe steroid geochemistry of a recent diatomaceous sediment from the Namibian shelf. Uceanol.Acta 5,365-378. SMITHD. J., EGLINTONG. and MORRISR. J. (1986) The lipid geochemistry of a recent sapropel and associati sediments from the Heflenic Outer Ridge. Eastern Mediterranean Sea. Phi!. Trans. Ro_v.Sot. London A-3i9,375-415. VOLKMANJ. K. (1986)A review of sterol markers for marine and tenigenous organic matter. Org. Geochem. 9,83-99. WAKEHAMS. G., GAGOSIANR. B., FARRINGTONJ. W. and CAN~JEL E. A. (1984) Sterenes in suspended particulate matter in the eastern tropical North Pacific. Nature 308, 840-843. WOLFF G. A., F'EAKMAN T. M. and MAXWELLJ. R. (1985) Semipreparative HPLC of some geologically interesting steroid hydrocarbons. J. High Rex Chrom.. Chrom. Commun. 8,695-696. WOLFFG. A., L&B N. A. and’M~xwn~~ J. R. (1986) The origin and fate of Qmethyl steroid hydrocarbons. I. D&genesis of 4-methyl sterenes. Geochim. Cosm~him. Acta SO,335-342.